This invention is generally in the area of genetically engineered urokinases, and in particular relates to an urokinase that is more resistant to inhibition by plasminogen activator inhibitors, and which can be selectively expressed and secreted from the mammary glands of a transgenic animal.
Plasminogen activators (PAs) catalyze the conversion, by limited proteolysis, of the circulating zymogen plasminogen to the broad spectrum protease plasmin, as reviewed, for example, by Collen, et al., Drugs 38: 346-388 (1989); Haber, et al., Science 243:51-56 (1989); Higgins and Bennett, Ann. Rev. Pharmacol. Toxicol. 30: 91-121 (1990); Mayer, Clin. Biochem. 23: 197-211 (1990); and Runge, et al., Circulation 79: 217-224 (1989).
Mammalian plasminogen activators are represented by two types of enzymes: tissue-type (t-PA) and urokinase-type (u-PA). Both enzymes can exist in single or two-chain (a- and b-chain) forms. Plasmin cleaves the activator at a single unique site, Lys158-Ile159 for u-PA and Arg275-Ile276 for t-PA. Cleavage does not alter the activator's molecular weight since the a and b-chains are held together by disulfide bonds. The single-chain form of t-PA (sct-PA) is an active enzyme, whereas the similar form of u-PA (scu-PA) is an inactive zymogen.
u-PA consists of an E-domain (Ser1-Asp45), Kringle-1 (Cys50-Cys131), and P-domain (Ile159-Leu411) containing the catalytic triad His204, Asp255, Ser356. The u-PA a-chain consists of the E-domain and Kringle-1. The E-domain of u-PA has been shown to function as a receptor-binding site by Appella, et al., J. Biol. Chem. 262:4437-4440 (1987).
To prevent systemic activation of plasminogen, the regulation of PA activity involves plasminogen activator inhibitors (PAIs). PAI-1 (originally designated endothelial type by Loskutoff and Edgington, J. Biol. Chem. 256: 4142-4145 (1981)), PAI-2 (originally designated placental type by Kawano, et al., Nature 217: 253-254 (1968)), and PAI-3 (originally designated urine type by Stump, et al., J. Biol. Chem. 261: 12759-12766 (1986)) are members of the serpin (serine protease inhibitor) superfamily. The inhibitors appear to function like other serine protease inhibitors, forming inactive covalent complexes with the target protease. PAI-1 rapidly inactivates both t-PA and u-PA. It is the major PAI of plasma, except during pregnancy. PAI-2 reacts more readily with u-PA than t-PA. PAI-2 is undetectable in the plasma of men and nonpregnant women, but rises to very high levels during late pregnancy.
The gene for urokinase has been characterized, and the protein produced, using recombinant techniques, as well as isolated from urine. Expression and secretion in the milk of transgenic animals has also been proposed, as described in U.S. Pat. No. 4,873,316 to Meade, et al.
For some time, u-PA, t-PA, and another structurally unrelated plasminogen activator, streptokinase, have been used as fibrinolytic therapeutic agents. The therapeutic goal is to dissolve heart attack-causing clots before they cause permanent damage to heart muscle, usually within four to six hours of the clot formation.
There are clinical advantages and disadvantages for each type of PA. Currently, streptokinase is the most affordable. However, t-PA theoretically has one major advantage over streptokinase. When t-PA binds fibrin, the Michaelis constant for the activation of plasminogen decreases by 100 fold, down to the in vivo concentration of plasminogen, as described by Hoylaerts, et al., J. Biol. Chem. 257: 2912-2919 (1982). t-PA therefore functions subsequent to binding fibrin in the clot. This concentrates the activated plasmin where it is most needed. Streptokinase degrades not just fibrin, but also fibrinogen, depleting the blood's normal clot-forming ability. Further unlike streptokinase, u-PA and t-PA are direct activators of plasminogen, can be highly purified, and are not bacterial in origin and therefore do not produce a reaction.
One therapeutic disadvantage of t-PA and u-PA is that they are rapidly inactivated by plasma PAIs, particularly PAI-1, following administration of the therapeutics. Recombinant PA derivatives with resistance to inactivation could theoretically yield higher plasma PA levels over a longer time, with higher thrombolytic potency.
It is therefore an object of the present invention to provide a biologically active prourokinase/urokinase that is more resistant to inhibition by plasminogen activator inhibitors.
It is a further object of the present invention to provide vectors containing sequences encoding the resistant prourokinase/urokinase which can be used to incorporate the genes into a transgenic animal for tissue specific expression which is not harmful to the host animal, especially expression in high levels in the milk of a transgenic animal.
It is another object of the present invention to provide vectors encoding short sequences of prourokinase/urokinase, and methods of use thereof, for ease in altering binding sites for plasminogen activator inhibitors.